Iranian Journal of Pharmaceutical Research (2013), 12 (4): 897-902 Received: February 2012 Accepted: August 2013
Copyright © 2013 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
Failure of Intravenous Lipid Emulsion to Reduce Diazinon-induced Acute Toxicity: a Pilot Study in Rats Mohammad Moshiria, Maryam Vahabzadeha, Leila Etemadb and Hossein Hosseinzadehc* a
Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. bPharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. cPharmaceutical Research Center, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Abstract Diazinon (DZN) is a synthetic organophosphorus (OPs) insecticide widely used in agricultural and household applications. OPs, particularly DZN, are highly lipid soluble liquids. Intravenous lipid emulsion (ILE) has been shown to reduce toxicity caused by some lipid soluble agents. We evaluated the antidote effect of ILE on acute toxicity of DZN. Twentyfour Sprague-Dawley female rats weighting 200-250 g were treated orally with dose of 480 mg/ kg of DZN gavaged at the volume of 0.5 mL/kg. Thirty minutes after administration of DZN, two groups were treated by either ILE 10% (ILE10) or normal saline (NS) (16 mL/kg) (NS16) that were infused for the duration of 15 minutes. Another two groups were also treated by either ILE 20% (ILE20) or NS (10 mL/kg: NS10) as above. The changes in body weight, diarrhea score, muscular power, fasciculation, convulsions and mortality rate of the animals were all monitored immediately after infusions and then every 6 h up to 48 h. There was no significant difference in animals mean weight between different groups during the observation period. In addition, during the 48-hour observation we could not find any difference in muscular power and diarrhea score between groups of ILE20-NS10 and ILE10-NS16 in comparison with each other, and neither ILE 10% nor ILE %20 could not reduce mortality rate of animals or increase the survival time of rats. In conclusion, ILE seems to be unable to reverse DZN acute toxicity and it might be due to conversion of DZN to potent and less lipid soluble agent. Keywords: Diazinon; Intravenous lipid emulsion; Organophosphorus.
Introduction Intravenous lipid emulsion (ILE) (intralipid) has been shown to reduce toxicity caused by some lipid soluble drugs (1). In many studies ILE has been an effective antidote in various types of intoxicated animals by different groups of drugs such as local anesthetics (2, 3), * Corresponding author: E-mail: [email protected]
propranolol (4) and tricyclic antidepressants (5, 6) Furthermore, ILE has been able to decrease morbidity and mortality in some intoxicated patients (7, 8). However, it was not completely effective in some poisoning such as clomipramine and paroxon (9-11). Diazinon (DZN) is a synthetic organophosphorus (OPs) insecticide widely used in agricultural, commercial, and household applications. Diazinon was classified as a class II ‘moderately hazardous’ pesticide by The World
Moshiri M et al. / IJPR (2013), 12 (4): 897-902
washed before each trial in order to prevent pheromonal-induced stress or cross-infection. Subsequently, a 25G plastic vein cannula was inserted in lateral tail vein under aseptic condition and attached to the animal tail properly. Thirty minutes after ingesting DZN, two groups were treated by either ILE 10% (ILE10) or Normal saline (16 mL/kg) (NS16) that were infused for the duration of 15 min. Another two groups were also treated by either ILE 20% or NS (10 mL/kg) (ILE 20 and NS 10 groups) which were infused during 15 min. By the end of infusions, (ILE or NS) animals were placed in separate cages with easy access to food and water. The changes in body weight, diarrhea score, muscular power, presence of fasciculation, and convulsions and mortality rate of the animals were all monitored immediately after infusions and then every 6 hours up to 48 h. Diarrhea was scored as follow: 0 = normal; 1 = loose stool; and 3 = defecation incontinence (22). The muscular powers of animals were evaluated using modified De Bleecker scoring- Grade 4: normal mobility; Grade 3: ataxic gait; Grade 2: stretch movements after tail stimulation; Grade 1: standing after tail stimulation; Grade 0: no voluntary movements after tail stimulation, four limb paralysis (23). All animals were evaluated by an individual unaware of the groups.
Health Organization (WHO) (12). There are approximately 3 million pesticide poisonings world-wide resulting in 220,000 deaths every year (13) and pesticide poisons are one of the main causes of poisonings (14, 15), or poisonrelated death in Iran (16). Diazinon as other OPs, act by interfering with nervous system function (17) Atropine and oximes are antidotes against acute toxicity of DZN (12) and some material such as saffron have antidotal effect against its sub acute toxicity (18, 19). OPs, particularly DZN, are highly lipid soluble liquids (20). DZN is hydrolyzed by water and some products which are more toxic than DZN itself (21). Some products of soybean oil are used as stabilizers, react with water preventing the hydrolysis of DZN, and subsequently form products that are more toxic. This mechanism, which has been also used by manufacturers, reduces DZN toxicity and enhances its median lethal dose (LD50) (21). We evaluated the antidotal effect of ILE (intralipid) on toxicity of organophosphate DZN, which is considered as a lipid-soluble substance. Experimental Animals and preparations: Twenty-four non-pregnant Sprague-Dawley female rats weighting 200-250 g were housed in cages with a 12 h light/dark cycle and easy access to food and water. Animals were divided into four groups of consisting six rats. Since DZN (BazodinR, Cyngenta, purity 96%) is poorly water soluble, it was dissolved in corn oil (Sigma) to gavage to animal. We also used ILE 10% (Lipovenoes® 10% , Germany ) or ILE20% (intralipid emulsion [Fresenius Kabi AB] Spain) or normal saline (NS) for intravenous treatment .
Statistics Results are expressed as mean ± standard deviations and were analyzed with T non-paired test and Mann–Whitney test by using Spss11.5 software. Statistical significance was set at P < 0.05 for all tests. Results At the beginning of the experiment, there was no significant difference in animals mean weight between different groups and it either showed insignificant changes during the further observation (Figurs 1 and 2). In addition, during the 48 h observation we could not find any difference in muscular power between groups of ILE20-NS10 and ILE10 - NS16 in comparison with each other (Figure 3 and 4). Moreover, there were no alterations in
Evaluation of ILE on DZN toxicity All of the animals were gavaged with dose of 480 mg/kg of DZN which was dissolved in corn oil at the gavaged volume of 0.5 mL/ kg. Rats were food-deprived overnight before the experiment in order to reduce the possible interaction between food and the toxin. Appropriate rodent restrainers were used to limit animal movements. The devices had been 898
Effect of ILE on Diazinon Toxicity Effect of ILE on Diazinon Toxicity
Table 1. comparing of mortality rate and means of survival times of four groups. Table 1. comparing of mortality rate and means survival times of fourp-VALUE groups. normal saline of 16cc ILE 10%
ILE 20%
p-VALUE
means of survival time (hours)
normal 33.0saline ± 5.3 16cc
ILE±10% 34.0 6.1
p-VALUE 0.91 (NS)
normal saline 10cc normal 35.0saline ± 5.9 10cc
ILE±20% 37.0 6.8
p-VALUE 0.83 (NS)
means ofrate survival time (hours) mortality
33.0 ± 5.3 66.67%
34.0 ± 6.1 66.67%
0.91(NS) (NS) 1.00
35.0 ± 5.9 66.67%
37.0 ± 6.8 50.00%
0.83(NS) (NS) 1.00
mortality=rate 66.67% 66.67%lipid emulsion 1.00 (NS)20%, numbers 66.67% 50.00% ILE10% intravenous lipid emulsion 10%, ILE20% = intravenous of animals in each group at 01.00 h = (NS) 6 ILE10% = intravenous lipid emulsion 10%, ILE20% = intravenous lipid emulsion 20%, numbers of animals in each group at 0 h = 6
Moreover, there were no alterations in diarrhea score score among among all all animal animal groups. groups. Thus, Thus, diarrhea comparingto tonormal normalsaline, saline,both bothconcentrations concentrations comparing of ILE ILE seemed seemed to to be be unable unable to to reduce reduce diarrhea diarrhea of score in poisoned animals (Figures 5 and 6)and and score in poisoned animals (Figures 5 and 6) neither ILE 10% nor ILE %20 could not reduce neither ILE 10% nor ILE %20 could not reduce the mortality mortality rate rate of of animals animals or or increase increase the the the survivaltime timeof ofrats rats(Table (Table1). 1). survival The incidence incidence of of fasciculation fasciculation between between four four The groups was insignificant during different times groups was insignificant during different times of observation. observation. Convulsion Convulsion did did not not occur occur in in any any of ofthe theanimals. animals. of Discussion Discussion Since2006 2006when whenthe thefirst firstcase casewas wasreported reportedto to Since be rescued by ILE (24), ILE has been evaluated be rescued by ILE (24), ILE has been evaluated asantidote antidotein inseveral severalkinds kindsof ofdrugs drugstoxicity, toxicity,such such as as local local anesthetics, anesthetics, tricyclic tricyclic antidepressants antidepressants as (1, 10, 10, 25), 25), propranolol propranolol (4), (4), atenolol atenolol (26), (26), and and (1, numerous other compounds. All compounds, numerous other drugs. All of the drugs, which whichsucceeded ILE has decreased their toxicity, high ILE in decreasing their have toxicity, have high lipid-solubility properties. The most important mechanism of action which has been suggested for antidote effect of ILE is “Lipid sink
theory” (27-29). Based on such theory, ILE acts lipid-solubility properties. The most important as a third compartment in blood that lipophilic mechanismcan of action which suggested substances be drawn intohasthebeen “lipid sink”, for antidote of ILE is “Lipid sinkbetween theory” resulting in effect a concentration gradient such theory, ILE actstoxin as a (27-29). tissue andBased blood.on This phenomenon causes as a new compartment in blood that lipophilic or drug to distance from the target tissues into substances can ofbeILE drawn the “lipid sink” (30).into the “lipid sink”, resulting a concentration between OPs andinespecially DZN aregradient extremely lipidtissue and blood. This phenomenon causes toxin soluble toxins (31), and according to the “lipid or drug to distance from the target tissues into sink” theory and the hydrolysis-inhibitory effect theILE “lipid sink” ILE (30). of (21), it is of supposed to reduce the toxicity OPs and especially DZN are extremely of DZN. Nevertheless, we failed to confirmlipidthis soluble toxins (31), and according to the “lipid hypothesis, likewise the research by Bania on sink” theory and on theanother hydrolysis-inhibitory the effect of ILE OP, Paraoxon effect (11). of ILEconcentrations (21), it is supposed to reduce Both of ILE (10% the andtoxicity 20%) of DZN. Nevertheless, we failedrate to confirm this failed to either reduce mortality or increase hypothesis, likewise the research by Bania on the the survival time in comparison with normal effect of ILE on another OP, Paraoxon (11). Both saline. concentrations of ILE (10% and (AChE), 20%) failed OPs inhibit acetylcholinesterase the to either mortality rate system or increase the critical andreduce widespread nervous enzyme survival time in comparison with normal saline. (31, 32), which degrades the neurotransmitter OPs inhibitinto acetylcholinesterase (AChE), the acetylcholine choline and acetic acid, resulting in overstimulation of muscarinic and nicotinic receptors (20). The muscarinic overstimulation could induce muscarinic sings
Fig Fig 2)2)
Figure 1. Effect of ILE 10% (16 mL/kg intravenous lipid FIG 1) emulsion in compare NS16(16 (16 mL/kg normal salinelipid on Figure 1.10%) Effect of ILE 10% intravenous means of weight rats which were by diazion. emulsion 10%) inofcompare NS16 (16intoxicated mL/kg normal saline on Pmeans = NS. of numbers in each group at 0 h = 6byand at 48 weightofofanimals rats which were intoxicated diazion. hP=non-significant. =2. numbers of animals in each group at 0 h = 6 and at 48 h = 2 .
Figure 2. Effect of ILE 20% (10 mL/kg intravenous lipid emulsion in compare NS10 mL/ kg normal saline Figure 2.20%) Effect of ILE 20% (10(10 mL/kg intravenous lipid on means of weight of rats which were by diazion. emulsion 20%) in compare NS10 (10intoxicated mL/ kg normal saline P-value = NS. numbers each group at 0by h =diazion. 6 and on means of weight of of ratsanimals which in were intoxicated atP=48Non-significant. h = 2. numbers of animals in each group at 0 h = 6 and at 48 h = 2.
? 899
Moshiri M etMal.et/al. IJPR (2013), 12 (4): 897-902 Mohammad / IJPR (2013), 12 (4): ?-?
normal saline normal saline 16cc/Kg 16cc/Kg
3.0 3.0
3.0 3.0
ILE%10 ILE%10 16cc/Kg 16cc/Kg
2.5 2.5 diarrhea score diarrhea score
diarrhea scoring diarrhea scoring
2.5 2.5
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ILE20% ILE20% 10cc/Kg 10cc/Kg
2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5
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0 0
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evalution evalution times( times( hours) hours)
12 12
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24 24
30 30
36 36
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evaluation evaluation time time (( hours) hours)
FIG 3)
Figure 3. 3. Effect Effect of of ILE ILE 10% 10% (16 (16 mL/kg mL/kg intravenous intravenous lipid lipid Figure emulsion 10%) 10%) in in compare compare NS16 NS16 (16 (16 mL/kg mL/kg normal normal saline) saline) on on emulsion means of of muscle muscle power power score score of of rats rats which which were were intoxicated intoxicated by by means diazion. P-value P= Non-significant. numbers of animals in each diazion. = NS. numbers of animals in each groupgroup at 0 = 6 at and hat=06hand 48ath 48 =2.h =2.
Figure 4. 4. Effect Effect of of ILE ILE 20% 20% (10 (10 mL/kg mL/kg intravenous intravenous lipid lipid Figure emulsion 20%) 20%) in in compare compare NS10 NS10 (10 (10 mL/kg mL/kg normal normal saline) saline) on on emulsion FIG 4 means ofmuscle musclepower powerscore scoreofof rats which were intoxicated means of rats which were intoxicated by by diazion. P= Non-significant. ofinanimals in each diazion. P value = NS. numbers numbers of animals each group at 0 hat=48 6 and at 48 h = 2. 0group h = 6atand h = 2.
Neitherinhibitors muscarinicofnor nicotinic DZN weak AChE (33, signs 35). ofWhilst toxicity were improved by different phosphorothionate OPs undergoconcentration metabolic of ILE. activation (desulfatation) to their corresponding 4.5 4.5 normal saline normal saline 16cc/Kg 16cc/Kg The three main classes of OPs oxygen analogues (oxon), theyinsecticides become 4.0 4.0 ILE10% 16cc/Kg ILE10% 16cc/Kg are 3.5 phosphorothionates, phosphorodithioates, extremely more potent (100-folds) (33, 35, 3.5 and 3.0 phosphoroamidothiolates (33, 34). DZN 36). The oxon product of DZN, diazoxon, has 3.0 to phosphorothionate which are abelongs high affinity and potency toOPs, phosphorylate 2.5 2.5 weak inhibitors AChE (33,the35). Whilst the serine hydroxylofgroup within active site 2.0 2.0 phosphorothionate undergo metabolic of AChE (33). SomeOPs authors believe that DZN 1.5 1.5 activation (desulfatation) to their do not directly inhibits AChE andcorresponding must first be 1.0 1.0 oxygen analogues they and become metabolized to diazoxon(oxon), (33, 37). DZN other 0.5 0.5 extremely (100-folds) (33, 35, OPs rapidly more absorbpotent following oral administration 0.0 0.0 36). The oxon of DZN, has (32) and undergo 0 0 3 3 6product 6 12a high 12 18 18 24hepatic 24 30diazoxon, 30 36first42 36 42 pass 48 48 time of of ev ev aluation(hours) aluation(hours) metabolism (33) astime only 35% of oral dose will means of means of muscle muscle power power score score
critical andover widespread nervous system glands), enzyme (diarrhea, secretion of exocrine (31, 32), whichoverstimulation degrades the neurotransmitter and nicotinic could induce acetylcholine choline and acetic acid, nicotinic signs into (fasciculation, convulsion and resulting paralysis). in overstimulation of muscarinic muscles We evaluated the bodyand nicotinic (20). The weight changesreceptors as an indicator of muscarinic water loss overstimulation could induce muscarinic sings due to diarrhea and over secretion of exocrine (diarrhea,which over had secretion of exocrine glands), glands, dehydrated the animals. and nicotinic overstimulation could ofinduce Neither muscarinic nor nicotinic signs DZN nicotinicwere signs (fasciculation, convulsion and toxicity improved by different concentration muscles of ILE. paralysis). We evaluated the bodyweight changes as classes an indicator water loss The three main of OPsofinsecticides due to diarrhea and over secretion of exocrine are phosphorothionates, phosphorodithioates, glands, which had dehydrated animals. and phosphoroamidothiolates (33,the34). DZN belongs to phosphorothionate OPs, which are
FIG 5)
normal normal saline saline 16cc/Kg 16cc/Kg
4.0 4.0
3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0 0
3 3
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24 24
30 30
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3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0
0.0 0.0
48 48
FIG 5)
0 0
3 3
6 6
12 12 18 18 24 24 30 30 ev aluation ev aluation time( time( hours) hours)
36 36
42 42
48 48
Fig 6
Figure 6. 6. Effect Effect of of ILE ILE 20% 20% (10 (10 ml/kg ml/kg intravenous intravenous lipid lipid Figure emulsion 20%) 20%) in in compare compare NS10 NS10 (10 (10 ml/kg ml/kg normal normal saline) saline) emulsion on means means of of diarrhea diarrhea score score of of rats rats which which were were intoxicated intoxicated by by on diazion. PP=value Non-significant. numbers of animals in each diazion. = NS. numbers of animals in each groupgroup at 0 = 6 at and hat=06hand 48ath 48 = 2.h = 2.
Figure 5. 5. Effect Effect of of ILE ILE 10% 10% (16 (16 mL/kg mL/kg intravenous intravenous lipid lipid Figure emulsion 10%) 10%) in in compare compare NS16 NS16 (16 (16 mL/ mL/ kg kg normal normal saline) saline) emulsion on means means of of diarrhea diarrhea score score of rats rats which were intoxicated intoxicated by by on of which were normal saline normal saline 10cc/Kg 10cc/Kg 4.5 4.5 diazion. P-value P= Non-significant. numbers of animals in each diazion. = NS. numbers of animals in each groupgroup at 0 ILE20% 10cc/Kg ILE20% 10cc/Kg 4.0 = 6 at and hat=04.0 6hand 48ath 48 = 2h = 2 means of means of muscle muscle power power
3.5 3.5
0.5 0.5
time time of of ev ev aluation(hours) aluation(hours)
3.5 3.5 3.0 3.0 2.5 2.5
900 ?
2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0
ILE20% ILE20% 10cc/Kg 10cc/Kg
4.0 4.0
3.5 3.5
0.0 0.0
normal saline normal saline 10cc/Kg 10cc/Kg
4.5 4.5
ILE10% ILE10% 16cc/Kg 16cc/Kg
means of means of muscle muscle power power
means of means of muscle muscle power power score score
4.5 4.5
0 0
3 3
6 6
12 12
18 18
24 24
30 30
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48 48
Effect of ILE on Diazinon Toxicity
a high affinity and potency to phosphorylate the serine hydroxyl group within the active site of AChE (33). Some authors believe that DZN do not directly inhibits AChE and must first be metabolized to diazoxon (33, 37). DZN and other OPs rapidly absorb following oral administration (32) and undergo a high hepatic first pass metabolism (33) as only 35% of oral dose will be eventually bioavailable (33). The activation of DZN to diazoxon is mediated by cytochrome P450 primarily within the liver, although some extrahepatic metabolism, such as the brain, has been reported (33, 38) Diazoxon is not as much lipophilic as its parent substance, DZN, (39). So it has 10 times less affinity for lipid comparing with DZN (40). The Partition coefficients (n-octanol-water) of Diazoxon and DZN are 2.07 and 3.81 respectedly (41). Thus, diazoxon is less trapped in fat compartments than DZN (42). Therefore, the major part of DZN changes into a 1000-times more potent and 10-times less lipid soluble product, diazoxon, prior to access to blood circulation. AS mentioned above the most important mechanism for antidotal effect of ILE is the “Lipid Sink” mechanism (29, 43) ,which an intravascular lipid compartment would be formed while adding a large amount of lipids into the blood. Such repartitioning can distant xenobiotics from the site of toxicity into the blood and send them to the liver to be metabolized and detoxified (44, 45). In conclusion, ILE seems to be unable to reverse DZN acute toxicity, and it might be due to conversion of DZN to potent and less lipid soluble agent.
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K, Hodgson E, Karczmar AG, Padilla S, Pope CN, Richardson RJ, Saunders DR, Sheets LP, Sultatos LG and Wallace KB. Common mechanism of toxicity: a case study of organophosphorus pesticides. Toxicol. Sci. (1998) 41: 8-20. (35) Sidiropoulou E, Sachana M, Flaskos J, Harris W, Hargreaves AJ and Woldehiwet Z. Diazinon oxon interferes with differentiation of rat C6 glioma cells. Toxicol. inVitro. (2009) 23: 1548-1552. (36) Poorheidari G, Khodaei N, Sahraei M, Saberi A, Shahriary A, Noroozzadeh A, Pirzad G and Khoshbaten A. Praoxon-induced changes in the function of chicken biventer cervices nerve-muscle preparation and the reversal of such changes by pralidoxime. Iran. J. Pharm. Res. (2004) 3: 89-89. (37) Iverson F, Grant DL and Lacroix J. Diazinon metabolism in the dog. Bull. Environ. Cont. Toxicol. (1975) 13: 611-8. (38) Guengerich FP. Separation and purification of multiple forms of microsomal cytochrome P-450. Activities of different forms of cytochrome P-450 towards several compounds of environmental interest. J. Biol. Chem. (1977) 252: 3970-9. (39) Vale JA. Toxicokinetic and toxicodynamic aspects of organophosphorus (OP) insecticide poisoning. Toxicol. Lett. (1998) 102-103: 649-52. (40) Gearhart JM, Jepson GW, Clewell HJ, Andersen ME, and Conolly RB. Physiologically based pharmacokinetic model for the inhibition of acetylcholinesterase by organophosphate esters. Envi. Health Perspect. (1994) 102 Suppl 11: 51-60. (41) Noble A. Partition coefficients (n-octanol-water) for pesticides. J. Chromatogr. (1993) 642: 3-14. (42) Timchalk C and Ramesh CG. Physiologically based pharmacokinetic modeling of organophosphorus and carbamate pesticides. In: Gupta RC. Toxicology of Organophosphate and Carbamate Compounds. Academic Press, Burlington (2006) 103-125. (43) Weinberg G. Lipid infusion resuscitation for local anesthetic toxicity: proof of clinical efficacy. Anesthesiol. (2006) 105: 7-8. (44) Weinberg GL, Ripper R, Murphy P, Edelman LB, Hoffman W, Strichartz G and Feinstein DL. Lipid infusion accelerates removal of bupivacaine and recovery from bupivacaine toxicity in the isolated rat heart. Reg. Anesth. Pain. Med. (2006) 31: 296-303. (45) Stellpflug SJ, Harris CR, Engebretsen KM, Cole JB and Holger JS. Intentional overdose with cardiac arrest treated with intravenous fat emulsion and high-dose insulin. Clin. Toxicol. (2010) 48: 227-9. This article is available online at http://www.ijpr.ir
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Copyright © 2013 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
Failure of Intravenous Lipid Emulsion to Reduce Diazinon-induced Acute Toxicity: a Pilot Study in Rats Mohammad Moshiria, Maryam Vahabzadeha, Leila Etemadb and Hossein Hosseinzadehc* a
Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. bPharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. cPharmaceutical Research Center, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Abstract Diazinon (DZN) is a synthetic organophosphorus (OPs) insecticide widely used in agricultural and household applications. OPs, particularly DZN, are highly lipid soluble liquids. Intravenous lipid emulsion (ILE) has been shown to reduce toxicity caused by some lipid soluble agents. We evaluated the antidote effect of ILE on acute toxicity of DZN. Twentyfour Sprague-Dawley female rats weighting 200-250 g were treated orally with dose of 480 mg/ kg of DZN gavaged at the volume of 0.5 mL/kg. Thirty minutes after administration of DZN, two groups were treated by either ILE 10% (ILE10) or normal saline (NS) (16 mL/kg) (NS16) that were infused for the duration of 15 minutes. Another two groups were also treated by either ILE 20% (ILE20) or NS (10 mL/kg: NS10) as above. The changes in body weight, diarrhea score, muscular power, fasciculation, convulsions and mortality rate of the animals were all monitored immediately after infusions and then every 6 h up to 48 h. There was no significant difference in animals mean weight between different groups during the observation period. In addition, during the 48-hour observation we could not find any difference in muscular power and diarrhea score between groups of ILE20-NS10 and ILE10-NS16 in comparison with each other, and neither ILE 10% nor ILE %20 could not reduce mortality rate of animals or increase the survival time of rats. In conclusion, ILE seems to be unable to reverse DZN acute toxicity and it might be due to conversion of DZN to potent and less lipid soluble agent. Keywords: Diazinon; Intravenous lipid emulsion; Organophosphorus.
Introduction Intravenous lipid emulsion (ILE) (intralipid) has been shown to reduce toxicity caused by some lipid soluble drugs (1). In many studies ILE has been an effective antidote in various types of intoxicated animals by different groups of drugs such as local anesthetics (2, 3), * Corresponding author: E-mail: [email protected]
propranolol (4) and tricyclic antidepressants (5, 6) Furthermore, ILE has been able to decrease morbidity and mortality in some intoxicated patients (7, 8). However, it was not completely effective in some poisoning such as clomipramine and paroxon (9-11). Diazinon (DZN) is a synthetic organophosphorus (OPs) insecticide widely used in agricultural, commercial, and household applications. Diazinon was classified as a class II ‘moderately hazardous’ pesticide by The World
Moshiri M et al. / IJPR (2013), 12 (4): 897-902
washed before each trial in order to prevent pheromonal-induced stress or cross-infection. Subsequently, a 25G plastic vein cannula was inserted in lateral tail vein under aseptic condition and attached to the animal tail properly. Thirty minutes after ingesting DZN, two groups were treated by either ILE 10% (ILE10) or Normal saline (16 mL/kg) (NS16) that were infused for the duration of 15 min. Another two groups were also treated by either ILE 20% or NS (10 mL/kg) (ILE 20 and NS 10 groups) which were infused during 15 min. By the end of infusions, (ILE or NS) animals were placed in separate cages with easy access to food and water. The changes in body weight, diarrhea score, muscular power, presence of fasciculation, and convulsions and mortality rate of the animals were all monitored immediately after infusions and then every 6 hours up to 48 h. Diarrhea was scored as follow: 0 = normal; 1 = loose stool; and 3 = defecation incontinence (22). The muscular powers of animals were evaluated using modified De Bleecker scoring- Grade 4: normal mobility; Grade 3: ataxic gait; Grade 2: stretch movements after tail stimulation; Grade 1: standing after tail stimulation; Grade 0: no voluntary movements after tail stimulation, four limb paralysis (23). All animals were evaluated by an individual unaware of the groups.
Health Organization (WHO) (12). There are approximately 3 million pesticide poisonings world-wide resulting in 220,000 deaths every year (13) and pesticide poisons are one of the main causes of poisonings (14, 15), or poisonrelated death in Iran (16). Diazinon as other OPs, act by interfering with nervous system function (17) Atropine and oximes are antidotes against acute toxicity of DZN (12) and some material such as saffron have antidotal effect against its sub acute toxicity (18, 19). OPs, particularly DZN, are highly lipid soluble liquids (20). DZN is hydrolyzed by water and some products which are more toxic than DZN itself (21). Some products of soybean oil are used as stabilizers, react with water preventing the hydrolysis of DZN, and subsequently form products that are more toxic. This mechanism, which has been also used by manufacturers, reduces DZN toxicity and enhances its median lethal dose (LD50) (21). We evaluated the antidotal effect of ILE (intralipid) on toxicity of organophosphate DZN, which is considered as a lipid-soluble substance. Experimental Animals and preparations: Twenty-four non-pregnant Sprague-Dawley female rats weighting 200-250 g were housed in cages with a 12 h light/dark cycle and easy access to food and water. Animals were divided into four groups of consisting six rats. Since DZN (BazodinR, Cyngenta, purity 96%) is poorly water soluble, it was dissolved in corn oil (Sigma) to gavage to animal. We also used ILE 10% (Lipovenoes® 10% , Germany ) or ILE20% (intralipid emulsion [Fresenius Kabi AB] Spain) or normal saline (NS) for intravenous treatment .
Statistics Results are expressed as mean ± standard deviations and were analyzed with T non-paired test and Mann–Whitney test by using Spss11.5 software. Statistical significance was set at P < 0.05 for all tests. Results At the beginning of the experiment, there was no significant difference in animals mean weight between different groups and it either showed insignificant changes during the further observation (Figurs 1 and 2). In addition, during the 48 h observation we could not find any difference in muscular power between groups of ILE20-NS10 and ILE10 - NS16 in comparison with each other (Figure 3 and 4). Moreover, there were no alterations in
Evaluation of ILE on DZN toxicity All of the animals were gavaged with dose of 480 mg/kg of DZN which was dissolved in corn oil at the gavaged volume of 0.5 mL/ kg. Rats were food-deprived overnight before the experiment in order to reduce the possible interaction between food and the toxin. Appropriate rodent restrainers were used to limit animal movements. The devices had been 898
Effect of ILE on Diazinon Toxicity Effect of ILE on Diazinon Toxicity
Table 1. comparing of mortality rate and means of survival times of four groups. Table 1. comparing of mortality rate and means survival times of fourp-VALUE groups. normal saline of 16cc ILE 10%
ILE 20%
p-VALUE
means of survival time (hours)
normal 33.0saline ± 5.3 16cc
ILE±10% 34.0 6.1
p-VALUE 0.91 (NS)
normal saline 10cc normal 35.0saline ± 5.9 10cc
ILE±20% 37.0 6.8
p-VALUE 0.83 (NS)
means ofrate survival time (hours) mortality
33.0 ± 5.3 66.67%
34.0 ± 6.1 66.67%
0.91(NS) (NS) 1.00
35.0 ± 5.9 66.67%
37.0 ± 6.8 50.00%
0.83(NS) (NS) 1.00
mortality=rate 66.67% 66.67%lipid emulsion 1.00 (NS)20%, numbers 66.67% 50.00% ILE10% intravenous lipid emulsion 10%, ILE20% = intravenous of animals in each group at 01.00 h = (NS) 6 ILE10% = intravenous lipid emulsion 10%, ILE20% = intravenous lipid emulsion 20%, numbers of animals in each group at 0 h = 6
Moreover, there were no alterations in diarrhea score score among among all all animal animal groups. groups. Thus, Thus, diarrhea comparingto tonormal normalsaline, saline,both bothconcentrations concentrations comparing of ILE ILE seemed seemed to to be be unable unable to to reduce reduce diarrhea diarrhea of score in poisoned animals (Figures 5 and 6)and and score in poisoned animals (Figures 5 and 6) neither ILE 10% nor ILE %20 could not reduce neither ILE 10% nor ILE %20 could not reduce the mortality mortality rate rate of of animals animals or or increase increase the the the survivaltime timeof ofrats rats(Table (Table1). 1). survival The incidence incidence of of fasciculation fasciculation between between four four The groups was insignificant during different times groups was insignificant during different times of observation. observation. Convulsion Convulsion did did not not occur occur in in any any of ofthe theanimals. animals. of Discussion Discussion Since2006 2006when whenthe thefirst firstcase casewas wasreported reportedto to Since be rescued by ILE (24), ILE has been evaluated be rescued by ILE (24), ILE has been evaluated asantidote antidotein inseveral severalkinds kindsof ofdrugs drugstoxicity, toxicity,such such as as local local anesthetics, anesthetics, tricyclic tricyclic antidepressants antidepressants as (1, 10, 10, 25), 25), propranolol propranolol (4), (4), atenolol atenolol (26), (26), and and (1, numerous other compounds. All compounds, numerous other drugs. All of the drugs, which whichsucceeded ILE has decreased their toxicity, high ILE in decreasing their have toxicity, have high lipid-solubility properties. The most important mechanism of action which has been suggested for antidote effect of ILE is “Lipid sink
theory” (27-29). Based on such theory, ILE acts lipid-solubility properties. The most important as a third compartment in blood that lipophilic mechanismcan of action which suggested substances be drawn intohasthebeen “lipid sink”, for antidote of ILE is “Lipid sinkbetween theory” resulting in effect a concentration gradient such theory, ILE actstoxin as a (27-29). tissue andBased blood.on This phenomenon causes as a new compartment in blood that lipophilic or drug to distance from the target tissues into substances can ofbeILE drawn the “lipid sink” (30).into the “lipid sink”, resulting a concentration between OPs andinespecially DZN aregradient extremely lipidtissue and blood. This phenomenon causes toxin soluble toxins (31), and according to the “lipid or drug to distance from the target tissues into sink” theory and the hydrolysis-inhibitory effect theILE “lipid sink” ILE (30). of (21), it is of supposed to reduce the toxicity OPs and especially DZN are extremely of DZN. Nevertheless, we failed to confirmlipidthis soluble toxins (31), and according to the “lipid hypothesis, likewise the research by Bania on sink” theory and on theanother hydrolysis-inhibitory the effect of ILE OP, Paraoxon effect (11). of ILEconcentrations (21), it is supposed to reduce Both of ILE (10% the andtoxicity 20%) of DZN. Nevertheless, we failedrate to confirm this failed to either reduce mortality or increase hypothesis, likewise the research by Bania on the the survival time in comparison with normal effect of ILE on another OP, Paraoxon (11). Both saline. concentrations of ILE (10% and (AChE), 20%) failed OPs inhibit acetylcholinesterase the to either mortality rate system or increase the critical andreduce widespread nervous enzyme survival time in comparison with normal saline. (31, 32), which degrades the neurotransmitter OPs inhibitinto acetylcholinesterase (AChE), the acetylcholine choline and acetic acid, resulting in overstimulation of muscarinic and nicotinic receptors (20). The muscarinic overstimulation could induce muscarinic sings
Fig Fig 2)2)
Figure 1. Effect of ILE 10% (16 mL/kg intravenous lipid FIG 1) emulsion in compare NS16(16 (16 mL/kg normal salinelipid on Figure 1.10%) Effect of ILE 10% intravenous means of weight rats which were by diazion. emulsion 10%) inofcompare NS16 (16intoxicated mL/kg normal saline on Pmeans = NS. of numbers in each group at 0 h = 6byand at 48 weightofofanimals rats which were intoxicated diazion. hP=non-significant. =2. numbers of animals in each group at 0 h = 6 and at 48 h = 2 .
Figure 2. Effect of ILE 20% (10 mL/kg intravenous lipid emulsion in compare NS10 mL/ kg normal saline Figure 2.20%) Effect of ILE 20% (10(10 mL/kg intravenous lipid on means of weight of rats which were by diazion. emulsion 20%) in compare NS10 (10intoxicated mL/ kg normal saline P-value = NS. numbers each group at 0by h =diazion. 6 and on means of weight of of ratsanimals which in were intoxicated atP=48Non-significant. h = 2. numbers of animals in each group at 0 h = 6 and at 48 h = 2.
? 899
Moshiri M etMal.et/al. IJPR (2013), 12 (4): 897-902 Mohammad / IJPR (2013), 12 (4): ?-?
normal saline normal saline 16cc/Kg 16cc/Kg
3.0 3.0
3.0 3.0
ILE%10 ILE%10 16cc/Kg 16cc/Kg
2.5 2.5 diarrhea score diarrhea score
diarrhea scoring diarrhea scoring
2.5 2.5
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ILE20% ILE20% 10cc/Kg 10cc/Kg
2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5
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0 0
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evalution evalution times( times( hours) hours)
12 12
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24 24
30 30
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evaluation evaluation time time (( hours) hours)
FIG 3)
Figure 3. 3. Effect Effect of of ILE ILE 10% 10% (16 (16 mL/kg mL/kg intravenous intravenous lipid lipid Figure emulsion 10%) 10%) in in compare compare NS16 NS16 (16 (16 mL/kg mL/kg normal normal saline) saline) on on emulsion means of of muscle muscle power power score score of of rats rats which which were were intoxicated intoxicated by by means diazion. P-value P= Non-significant. numbers of animals in each diazion. = NS. numbers of animals in each groupgroup at 0 = 6 at and hat=06hand 48ath 48 =2.h =2.
Figure 4. 4. Effect Effect of of ILE ILE 20% 20% (10 (10 mL/kg mL/kg intravenous intravenous lipid lipid Figure emulsion 20%) 20%) in in compare compare NS10 NS10 (10 (10 mL/kg mL/kg normal normal saline) saline) on on emulsion FIG 4 means ofmuscle musclepower powerscore scoreofof rats which were intoxicated means of rats which were intoxicated by by diazion. P= Non-significant. ofinanimals in each diazion. P value = NS. numbers numbers of animals each group at 0 hat=48 6 and at 48 h = 2. 0group h = 6atand h = 2.
Neitherinhibitors muscarinicofnor nicotinic DZN weak AChE (33, signs 35). ofWhilst toxicity were improved by different phosphorothionate OPs undergoconcentration metabolic of ILE. activation (desulfatation) to their corresponding 4.5 4.5 normal saline normal saline 16cc/Kg 16cc/Kg The three main classes of OPs oxygen analogues (oxon), theyinsecticides become 4.0 4.0 ILE10% 16cc/Kg ILE10% 16cc/Kg are 3.5 phosphorothionates, phosphorodithioates, extremely more potent (100-folds) (33, 35, 3.5 and 3.0 phosphoroamidothiolates (33, 34). DZN 36). The oxon product of DZN, diazoxon, has 3.0 to phosphorothionate which are abelongs high affinity and potency toOPs, phosphorylate 2.5 2.5 weak inhibitors AChE (33,the35). Whilst the serine hydroxylofgroup within active site 2.0 2.0 phosphorothionate undergo metabolic of AChE (33). SomeOPs authors believe that DZN 1.5 1.5 activation (desulfatation) to their do not directly inhibits AChE andcorresponding must first be 1.0 1.0 oxygen analogues they and become metabolized to diazoxon(oxon), (33, 37). DZN other 0.5 0.5 extremely (100-folds) (33, 35, OPs rapidly more absorbpotent following oral administration 0.0 0.0 36). The oxon of DZN, has (32) and undergo 0 0 3 3 6product 6 12a high 12 18 18 24hepatic 24 30diazoxon, 30 36first42 36 42 pass 48 48 time of of ev ev aluation(hours) aluation(hours) metabolism (33) astime only 35% of oral dose will means of means of muscle muscle power power score score
critical andover widespread nervous system glands), enzyme (diarrhea, secretion of exocrine (31, 32), whichoverstimulation degrades the neurotransmitter and nicotinic could induce acetylcholine choline and acetic acid, nicotinic signs into (fasciculation, convulsion and resulting paralysis). in overstimulation of muscarinic muscles We evaluated the bodyand nicotinic (20). The weight changesreceptors as an indicator of muscarinic water loss overstimulation could induce muscarinic sings due to diarrhea and over secretion of exocrine (diarrhea,which over had secretion of exocrine glands), glands, dehydrated the animals. and nicotinic overstimulation could ofinduce Neither muscarinic nor nicotinic signs DZN nicotinicwere signs (fasciculation, convulsion and toxicity improved by different concentration muscles of ILE. paralysis). We evaluated the bodyweight changes as classes an indicator water loss The three main of OPsofinsecticides due to diarrhea and over secretion of exocrine are phosphorothionates, phosphorodithioates, glands, which had dehydrated animals. and phosphoroamidothiolates (33,the34). DZN belongs to phosphorothionate OPs, which are
FIG 5)
normal normal saline saline 16cc/Kg 16cc/Kg
4.0 4.0
3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0 0
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24 24
30 30
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3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0
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48 48
FIG 5)
0 0
3 3
6 6
12 12 18 18 24 24 30 30 ev aluation ev aluation time( time( hours) hours)
36 36
42 42
48 48
Fig 6
Figure 6. 6. Effect Effect of of ILE ILE 20% 20% (10 (10 ml/kg ml/kg intravenous intravenous lipid lipid Figure emulsion 20%) 20%) in in compare compare NS10 NS10 (10 (10 ml/kg ml/kg normal normal saline) saline) emulsion on means means of of diarrhea diarrhea score score of of rats rats which which were were intoxicated intoxicated by by on diazion. PP=value Non-significant. numbers of animals in each diazion. = NS. numbers of animals in each groupgroup at 0 = 6 at and hat=06hand 48ath 48 = 2.h = 2.
Figure 5. 5. Effect Effect of of ILE ILE 10% 10% (16 (16 mL/kg mL/kg intravenous intravenous lipid lipid Figure emulsion 10%) 10%) in in compare compare NS16 NS16 (16 (16 mL/ mL/ kg kg normal normal saline) saline) emulsion on means means of of diarrhea diarrhea score score of rats rats which were intoxicated intoxicated by by on of which were normal saline normal saline 10cc/Kg 10cc/Kg 4.5 4.5 diazion. P-value P= Non-significant. numbers of animals in each diazion. = NS. numbers of animals in each groupgroup at 0 ILE20% 10cc/Kg ILE20% 10cc/Kg 4.0 = 6 at and hat=04.0 6hand 48ath 48 = 2h = 2 means of means of muscle muscle power power
3.5 3.5
0.5 0.5
time time of of ev ev aluation(hours) aluation(hours)
3.5 3.5 3.0 3.0 2.5 2.5
900 ?
2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0
ILE20% ILE20% 10cc/Kg 10cc/Kg
4.0 4.0
3.5 3.5
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normal saline normal saline 10cc/Kg 10cc/Kg
4.5 4.5
ILE10% ILE10% 16cc/Kg 16cc/Kg
means of means of muscle muscle power power
means of means of muscle muscle power power score score
4.5 4.5
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Effect of ILE on Diazinon Toxicity
a high affinity and potency to phosphorylate the serine hydroxyl group within the active site of AChE (33). Some authors believe that DZN do not directly inhibits AChE and must first be metabolized to diazoxon (33, 37). DZN and other OPs rapidly absorb following oral administration (32) and undergo a high hepatic first pass metabolism (33) as only 35% of oral dose will be eventually bioavailable (33). The activation of DZN to diazoxon is mediated by cytochrome P450 primarily within the liver, although some extrahepatic metabolism, such as the brain, has been reported (33, 38) Diazoxon is not as much lipophilic as its parent substance, DZN, (39). So it has 10 times less affinity for lipid comparing with DZN (40). The Partition coefficients (n-octanol-water) of Diazoxon and DZN are 2.07 and 3.81 respectedly (41). Thus, diazoxon is less trapped in fat compartments than DZN (42). Therefore, the major part of DZN changes into a 1000-times more potent and 10-times less lipid soluble product, diazoxon, prior to access to blood circulation. AS mentioned above the most important mechanism for antidotal effect of ILE is the “Lipid Sink” mechanism (29, 43) ,which an intravascular lipid compartment would be formed while adding a large amount of lipids into the blood. Such repartitioning can distant xenobiotics from the site of toxicity into the blood and send them to the liver to be metabolized and detoxified (44, 45). In conclusion, ILE seems to be unable to reverse DZN acute toxicity, and it might be due to conversion of DZN to potent and less lipid soluble agent.
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